Proteomics Facility (Wistar)

Summary:

The Wistar Proteomics Facility provides mass spectrometry (MS) and sequence analysis of proteins and peptides at maximum sensitivity using state-of-the-art instruments and methods.

The most commonly used services are identifications of either purified proteins or complex protein mixtures, such as sub-proteomes or complete proteomes, using electrospray ionization tandem mass spectrometry (ESI MS/MS). Typically, either individual bands are excised from 1-D SDS gels, or the entire gel lane is analyzed by slicing it into uniform fractions followed by trypsin digestion and nanocapillary HPLC interfaced directly with hybrid ion trap mass spectrometry (Gel/LC-MS/MS). Data is analyzed and filtered to produce low false-positive rates. Several options are available for quantitatively comparing protein changes in related samples, and additional options will be implemented in the future.

Complementary services include reverse-phase microbore HPLC peptide mapping with UV detection and mass measurements of intact peptides and proteins using MALDI MS or ESI MS. Posttranslational modification (PTM) analyses including identifications of specific modified residues also are provided, although investigators should recognize that in most cases these studies are quite complex and require substantial effort. These studies, as well as analyses of complex protein mixtures, usually require preparation of custom sequence databases and/or custom data analyses, which can be provided by the facility as needed.

Services

"Purpose: Standard database searches from individual LC-MS/MS runs and filtering out low-confidence hits are included in the services described in LC-MS/MS protein identifications service. More extensive data analyses, including searching for PTMs or other non-standard modifications, combining multiple datasets from subproteome/proteome analyses, and quantitative comparisons of multiple samples either globally or for specific peptides, are available from the facility upon request. Other specialized applications, including using programs such as GPMAW, can be performed by facility staff upon request.

"Purpose: Some projects require specialized databases, such as multiple isoforms of a protein of interest or a recombinant protein that does not exactly match the sequence in the database due to cloning-related changes. These user-provided sequences are combined with sequences of expected or likely contaminants, some of which might be experiment-specific. Such sequences are used when searching for PTMs, etc.

Requirements: Purified intact protein in ESI compatible buffer such as 0.05% acetic acid. It must be free of detergents such as SDS, any salts, etc. Concentration of 10 pmol/µl preferred. Samples can be cleaned up and/or concentrated if needed for an additional charge.

Results: Printout of convoluted/deconvoluted spectra with mass of protein found and controls in PDF file format."

"Purpose: Identify as many proteins as possible in a subproteome or an entire proteome. Examples of subproteomes include analysis of a purified large macromolecular complex, a highly purified organelle, a phosphotyrosine sub-proteome, etc. Examples of proteomes include: a cell line, the secretome of a cell line grown in serum-free media, etc.

Requirements: An intact 1D SDS mini-gel (see In-gel protease digestion service for details and detection limits/expected results). Depending upon the number of fractions that are to be analyzed and the complexity of the sample, the optimal electrophoresis distance will vary. For example, if the sample is relatively simple (<100 proteins), the SDS gel should only be run until the bromophenol tracking dye reaches either 1.0 or 2.0 cm. It is strongly recommended that you consult with the Facility Manager before preparing the sample to be submitted. Include a protein standard on the gel that is separated from the sample lane by one to two blank lanes. Submit an image of the gel with bands/spots of interest and standard Molecular Weights clearly marked and a completed MS/MS submission form (PDF). Complete all entries on the submission form, including the species of sample origin, Molecular Weight, gel thickness, type, etc.

Results: A large number of high-confidence (>99%) identifications are expected where the proteins have been identified by two or more peptides, provided that the genome for the species analyzed is essentially complete. Integrating the protein identifications from multiple LC-MS/MS analyses requires extensive specialized computational analysis that will vary depending upon proteome complexity. Further extensive critical evaluation and data refinement by the investigator generally is recommended prior to publication of results."

"Purpose: Enzymatic digestion of proteins in polyacrylamide gels is the preliminary procedure for multiple alternative services including LC-MS/MS, Gel/LC-MS/MS, Peptide mapping, and PTM analysis. The micro digest used here has been developed to minimize volumes and maximize peptide yields which are typically >80%. Porcine-modified trypsin, the protease of choice, cleaves at the carboxyl side of lysine and arginine residues. For special applications such as analysis of PTMs, other proteases also can be used.

Requirements: Submit an intact 1D or 2D gel stained with colloidal Coomassie® (preferred) or MS-compatible silver stain that was prepared following our "Sequence-quality SDS-gel guidelines." For simple protein identification using LC-MS/MS, as little as 5 femtomoles of the desired protein in a single mini-gel band or 2D gel spot typically will yield identification of at least the most abundant protein present; that is, even barely detectable silver stained bands will result in identifications, although sequence coverage will be quite limited when near the limit of detection. After staining/destaining gel, rinse it for one hour with high purity water, seal in plastic bag, photograph or scan the gel, and mark the desired protein band(s) on the gel image (annotated image in PowerPoint preferred). Store gel at 4°C until the entire gel is delivered to the facility.

Results: Small-volume tryptic digest of purified protein or complex protein mixtures for further characterization via LC-MS/MS, etc. Purified proteins invariably can be identified from barely detectable Colloidal Coomassie® bands/spots, and most barely detectable silver-stained bands/spots usually can be identified with limited sequence coverage. Gel slices with intense Colloidal Coomassie® staining from complex proteomes can yield confident identification of >100 proteins for each slice, and up to several thousand proteins from an entire gel lane. "

"Purpose: Identification of proteins at high sensitivity from tryptic peptides (or peptides produced by alternative proteases), or identification of locations of chemical modifications or PTMs on purified proteins. Nano-capillary reverse-phase HPLC is interfaced directly with hybrid linear ion trap mass spectrometers (LC-MS/MS) for maximum sensitivity. Proteins are identified by comparing MS/MS spectra to sequence databases, typically using SEQUEST®. For reliable identifications, the exact protein sequence must be in the database. Highly homologous proteins sometimes can be identified if a substantial portion of the tryptic peptides have identical sequences.

Requirements: An intact 1D or 2D gel (see #1 above for details and detection limits/expected results). Include an image of the gel with bands/spots of interest clearly marked and a completed "MS/MS Submission Form." Complete all entries on the submission form, including the species of sample origin, Molecular Weight, gel thickness, type, etc. Requires service in-gel protease digestion.

Results: High-confidence (>99%) identifications are expected for proteins identified by two or more peptides, provided that the protein or a very close relative is in the sequence database used for the search. However, it should be noted that in a substantial number of cases, the MS/MS matches to peptide sequences for an identified protein actually only define a family of homologous proteins. That is, there may be insufficient sequence coverage to unambiguously define a single protein. Hence, in a multi-species search, the protein from an incorrect species may be listed or an isoform or splice variant may be listed that is homologous but not identical to the isoform present in the analyzed sample.

The SEQUEST® output from the database search consists of a list in PDF file format of peptide sequences matched to protein(s). For complex protein mixtures, the list of peptide identifications may not be listed to reduce report length, but a full list of peptides can readily be provided. It should be noted that all identifications of peptides and proteins based on MS/MS data are identified based on correlation of observed peptide masses and fragmentation patterns with those predicted from sequences in the database. Even when data is stringently filtered, there is a very small but finite possibility that any individual peptide identification is incorrect."

"Purpose: Determine masses of purified peptides or proteins in solution. MALDI mass analysis is feasible over a wide mass range and can help identify the extent of post-translational modifications such as oxidation, glycosylation, and phosphorylation. It is the method of choice to check integrity of synthetic peptides, peptides from fractions of HPLC separations, and integrity of recombinant proteins. While some salts and buffers are tolerated, it may be necessary to 'clean up' the sample prior to analysis if buffer components suppress the mass signal.

Requirements: Purified peptides for routine analysis should be between 0.5-5 pmoles/µl in 0.1% TFA (sample also may contain up to 50% acetonitrile). Proteins should be 10 pmoles/µl, preferably in 20 mM NH4HCO3. It is especially important to avoid the following: Phosphate buffer >20 mM, most detergents >0.1% or SDS >0.01%, alkali metal salts >1M, glycerol >1%, Tris buffer >10 mM, guanidine or urea >1 M, or sodium azide >1 mM.

Results: Printouts of the masses found and a printout of the standard used for the calibration of the instrument in PDF file format."

"This service is analogous to Gel/LC-MS/MS except that the subproteome or proteome is digested with a protease in solution rather than separation on a gel, and the digest then is fractionated by ion exchange chromatography prior to LC-MS/MS analysis as described above. This method typically yields less protein identifications and sequence coverage compared with Gel/LC-MS/MS when the same number of fractions and LC-MS/MS runs are used. In addition, there is no information concerning approximate Molecular Weight of the identified proteins. Hence, Gel/LC-MS/MS is recommended for most subproteome and proteome analyses. Nonetheless, MudPIT can be used as needed in special situations."

"Purpose: Identification and location of post-translational modifications such as phosphorylation, acetylation, etc. This can be accomplished using several services such as in-gel digestion, MALDI MS, LC-MS/MS, and RP-HPLC mapping, depending upon the type of project.

Requirements: Project dependent (inquire with Facility Manager). Typically, an intact 1D or 2D gel stained with Coomassie® Blue R-250 or colloidal Coomassie® that was prepared following our "Sequence-quality SDS-gel guidelines," same as for LC-MS/MS.

Results: Printouts of description of post-translational modifications found and supporting data in PDF file format."

"Purpose: High-resolution, reversed-phase, chromatographic separation of in-gel tryptic digest for comparative mapping of two or more gel bands. Comparative mapping is the most quantitative method for detecting differences and similarities between different bands on a gel such as chemical or post-translational modifications, proteolytic processing, etc.

Requirements: Tryptic digest performed in this facility of gel bands containing at least 10 pmoles of target protein(s).

Software

"SEQUEST correlates uninterpreted tandem mass spectra of peptides with amino acid sequences from protein and nucleotide databases. SEQUEST will determine the amino acid sequence and thus the protein(s) and organism(s) that correspond to the mass spectrum being analyzed."